Altered vascularity and deregulated metabolism are two important cancer hallmarks. Interest in therapeutically exploiting these functional endpoints continues to grow, since metabolism and vasculature significantly impact a tumor?s fate. There is growing demand for systems-level technologies that measure tumor metabolism within an intact microenvironment to inform strategies to prevent resistance, recurrence and metastases. Vascular oxygen saturation (SO2) and total hemoglobin concentration ([THb]) within the tumor microenvironment influence metabolism by affecting substrate availability; by the same token, the nature of metabolism affects these vascular parameters by affecting substrate demand. Considering the importance of metabolism and vascularity to cancer biology there are surprisingly few techniques available to provide a systems-level approach to measure metabolism and the associated vasculature in vivo. There is a significant unmet preclinical need for simultaneous measurement of vascularity and metabolism of tumors in vivo for pharmacology research. Our group has proposed a plan to develop, validate, and commercialize a portable, adjunct optical spectroscopy device that can quantitatively measure vascular and metabolic endpoints including oxygen saturation, total hemoglobin concentration, glucose uptake and mitochondrial membrane potential in tumors in vivo. Zenalux Biomedical has previously demonstrated that quantitative diffuse reflectance spectroscopy (ZenascopeTM) can be used to assess relevant vascular endpoints in a variety of cancers. Independently, the Ramanujam lab has demonstrated with both in vivo microscopy of animal models and in vivo fluorescence spectroscopy of phantoms the ability to simultaneously measure key endpoints of metabolism using the fluorophores 2-NBDG (glucose uptake) and TMRE (mitochondrial membrane potential). We have detailed a plan in this Phase I STTR proposal to extend the capability of our existing Zenascope? to a multi- parametric spectroscopy system capable of providing accurate and precise analysis of metabolism and functional vascular endpoints of solid tumors in small animals. The performance of the portable Zenascope? ZF-1 system will be optimized against the gold-standard research grade system using tissue-mimicking fluorescence phantoms. The simultaneous capture of 2-NBDG and TMRE will be optimized and extensively validated in vivo in 4T1, 4T07 and 67NR murine breast cancer tumors using the proposed Zenascope? ZF-1 system. Further the Zenascope? ZF-1 measured optical endpoints will be validated against metabolic features independently measured by well- established laboratory techniques (Seahorse Assay and Metabolomics). The primary deliverables will be: (1) a portable spectroscopy platform (Zenascope? ZF-1) that is able to simultaneously capture multiple vascular and metabolic endpoints; and (2) an optimized and validated protocol for simultaneous spectroscopy of glucose uptake and mitochondrial oxidative phosphorylation in solid tumors in vivo. The resulting ZF-1 will fill the critical need for systems-level tools to measure cancer metabolism in small animal models, and will enable investigations ranging from fundamental studies of cancer signaling pathways to clinical patient-derived xenograft studies to test targeted agents for personalized therapy.